1. Field of the Invention
The present invention relates to an improvement in the field of cytology and more particularly a microscopic method of supravital blood analysis by which under normal white light illumination of a microscopic field optical differentiation, identification, comparisons and ennumeration of each of the five white blood cells is made possible by use of a single pure dye without fixation and more accurately and rapidly than heretofore by manual or automated differential leukocyte counters.
2. Description of the Prior Art
Ehrlich made biological elements more readily and easily recognized under microscopic examination and for photographic observation by use of dye stains (aniline dyes) to identify certain white blood cells. Ehrlich was the first to note that some dyes were metachromatic, observing that the staining of the cell causes the cell to take on a color different than that of the stain or expected color from the stain. Basophils, for example, were observed to take on a color different from the stain. Other histological specimens other than blood cells have also been reported to stain in a plurality of identifiably different colors.
A review of the state of the art indicates it is almost universal practice, before staining (which ordinarily uses a plurality of chemically differing dyestuffs in admixture) to employ a fixative procedure which may require up to an half hour treatment before the biological specimen is subjected to dye stain. Fixatives are generally preservatives and denaturants that often interfere with the sensitivity of the dye sorption. Illustratively, fixatives include formaldehyde both as liquid and vapor, absolute alcohols (methyl), picroformal, etc. Very often living cells do not stain using vital dyes and fixatives have been essential to staining the specimens. Cytochemistry includes considerable information on techniques developed to assure reproducible staining of blood cells. Many essential additives are normally unstable and deteriorate rapidly, thus making cellular identification difficult and in some instances unreliable. Dr. Thomas E. Necheles has observed in relation to leukocyte analysis That this "system has undergone little or no change in fifty years".
Dye staining does serve, however, as a means of discernment of otherwise undiscernable detail of conferring a color reaction on cells and their stainable components; metabolic, functional or pathological.
United States hospitals began leukocyte counting in the early 1900's, using the count as indicia as to whether emergency surgery was necessary, for example. In the U.S. alone, more than half a million differential counts are performed every day, most of them by manual methods. It is important that total white cell counts and differential cell counts be performed and reported without delay. Time is of essence and providing required analysis more rapidly is a desideratum.
The value of leukocyte counting having been established, the demand for rapid blood analysis has developed so that beginning about 1950 with the work of Mellors and Papaincolaou (1952) development of automated differential leukocyte counting instrumentation means had developed into a plurality of instruments by 1980. The "CYDAK" unit was early used to investigate the feasability of blood cell classification which pointed up the importance of specialized staining procedures and features were extracted from optical density histograms of each cell image. The procedure established that cells could be differentiated into four of the five classes of leukocytes, namely; neutrophils, eosinophils, lymphocytes and monocytes. Young (1969) published results on an automated classification of five cell classes and Bacus in 1971 extended the differentiation.
However, it is understood that automated differential systems presently rely upon multiple dye usage and dye degradation systems or indirect fluorescent measurement using fluorescent dyes. The latter are described by Kleineman in U.S. Pat. Nos. 3,916,205 and 4,146,604 which disclosure is included herein by reference and indicates the present state of the art in some measure. Here again it will be noted that fixatives were employed as is standard practice in the method of the patentee.
In the prior art staining of blood it has been observed that it is practice to use two or more stains in combination (Romanowski, Giemsa and Wright stains). These methods are difficult in practice to provide quality control. The methods require standardization in preparation of each dye stain component as well as in the method of specimen staining. In development of successful automated leukocyte counters, reproducibility of staining is even more important to verifiable analysis.
"LARC" stainer (used in commercial automated differential leukocyte counter) is reported (Mogler 1973) to be a mixture of some ten thiazine dyes, eosin Y and 2.sup.1, 4.sup.1, 5.sup.1 tribromofluorescein (P. N. Marshall). Present art stains most often are in fixative alcoholic solutions and employ two or more stains in combination. Accurate analysis of vital blood staining is made most difficult. With the difficulty presented in the controlled oxidation of methylene blue essential to Romanowski stains, for example, the problems of quality control of the added ten individually different dye stains as are used in combination become awesome.
It has been recognized in the art that the wide-spread standardization and adoption of a limited number of stains would ensure greater accuracy and reproducibility in cytological studies. Serious introduction of artifacts have been observed by use of fixatives and cause difficulty in interpretation and misinterpretation in leukocyte differentiation and ennumeration. pH adjustments, heavy metal cations have been reported to prevent cytochemical tests from working in the expected manner. Some dyes, particularly azo dyes, are noted to demonstrate non-specific precipitation around cells; other degenerative changes in fixed blood samples include vacuoles, clover-leafing of nuclei, distortion cell shapes and smudges and interference with ideal staining. The importance of performing differential counts on as near living cells in the shortest possible time in order to obtain optimally useful and valuable blood cell analyses has been recognized. Alcoholic dye solutions interfere with supravital staining. So far as is known, freshly prepared water soluble stains exhibit a minimum denaturant effect upon supravital blood during examination. All dyestuffs are more or less toxic to the blood cells, but some are more so than others. It is material that the cells under examination remain living as long as possible. Rapidity of staining obviously shortens the exposure time, thus allowing greater opportunity to examine leukocyte cells before all vitality is lost. Automated differential leukocyte counting in less minutes is sought for.
Studies and review of the prior art of performing microscopic blood analyses and disease diagnosis has indicated it is not unusual for pathologists to warm the dye and the blood speciment to body temperatures (about 37.degree. C.) before contact. Dr. Sabin had a "warm box" to insure temperature control.
It has also been noted that some dyes used in the prior art are quite temperature sensitive. The literature reports that cresylecht violet is not an operative stain above 30.degree. C. and the most favorable temperature for neutral red is about 32.degree. C. Neutral red gives no appreciable differential color staining. It is considered important for the purposes of this method as disclosed herein that the dyestuff be useful to stain leukocytes at temperatures as high as 37.degree. and no difficulty has been observed with the select dyes to temperatures of about 40.degree. C.
This invention has been found to be limited to basic cationic dyestuffs, and that they are as a class, relatively few in number in comparison to all the many classification of known dyestuffs. The Colour Index for (1956-63) lists some 3000 synthetic organic dyestuffs. Of these only 190 are cationic. Out of one relatively extensive available catalog listing of basic cationic dyes there were sixty-one listed species tested in the present inventive use. Of the total tested, three were found useful for the purposes and within purview of this invention. In another survey of eighteen basic quaternary dyes in a listed public dye offering, only one was found to stain one species of leukocyte metachromatically.
Insofar as is known, the prior art fails to disclose basic cationic quaternary dyestuffs effective to selectively stain species of leukocytes, to be effective metachromatically and at temperatures of 37.degree. C.-40.degree. C. The prior art discloses no specific dyes used to distinguish monocytes instantly from others of the leukocyte species. The prior art discloses no simple method for identification and counting of lymphocytes.